Epidemiology and Clinical Outcomes of HIV Infection in South-Central China: A Retrospective Study From 2003 to 2018

Objective HIV epidemiology in South-Central China is rarely reported. This study aims to characterize epidemiological and clinical features of HIV-infected patients in Hunan Province, located in South-Central China, for better management of HIV infections. Methods This retrospective study retrieved multi-center records of laboratory-confirmed HIV-infected patients in Hunan province. Information on HIV-associated mortality and antiretroviral therapies was also collected. Results Among 34,297 patients diagnosed with HIV infections from 2003 to 2018, 73.9% were males, 41.3% were older adults (≥50 years), and 71.2% were infected by heterosexual transmission. Despite a slow growth of new HIV infections in the overall population, annual percentages of HIV infections increased in older males (85.3% through heterosexual transmission) and young patients <30 years (39.9% through homosexual transmission). At baseline, serum levels of CD4+ T-cell counts were lower in older adults (191.0 cells/μl) than in young patients (294.6 cells/μl, p-value < 0.0001). A large proportion (47.2%, N = 16,165) of HIV-infected patients had advanced HIV disease (CD4+ T-cell counts < 200 cells/μl) from 2003 to 2018. All-cause mortality (57.0% due to AIDS-related illnesses) was reported among 4411 HIV-infected patients, including 2619 older adults. The 10-year survival rate was significantly lower in elderly males than in other patients (59.0 vs. 78.4%, p-value < 0.05). Conclusions Elderly males are prone to HIV infections with a high risk of HIV-associated fatality. Our findings support early prevention and critical care for elderly populations to control HIV infections.


INTRODUCTION
Human immunodeficiency virus (HIV) infection causes progressive damage to the immune system, characterized by massive depletion of CD4+ T-cells, sustained immune activation, and systemic inflammation (1). As of today, more than 37 million patients are living with HIV globally and ∼1.1 million cases are living in China (http://www.unaids.org). Since the introduction of antiretroviral therapies (ART) in the 1980s (2,3), the mortality among people living with HIV has decreased significantly (4) and the life expectancy of HIV-infected patients has improved dramatically (5,6). Most antiretroviral regimens consist of three oral compounds, including two nucleoside reverse transcriptase inhibitors (NRTIs) plus either one non-nucleoside reverse transcriptase inhibitor (NNRTI), one integrase inhibitor, or one protease inhibitor boosted by ritonavir or cobicistat (7)(8)(9). Although many highly active antiretroviral therapies have been approved in the past decades (3,9), an effective drug or vaccine remains unavailable to "cure" HIV infections (10). To curb HIV infections, it remains important for epidemiological studies to identify high-risk populations in different countries and regions.
Since the first report of HIV infection in China in 1989 (11), HIV has spread across the country in the past decades. In 2018, 64,170 patients and 18,780 HIV-related deaths were reported in China (12). The overall incidence of HIV in China was not high but on the rise over time, and the fatality rate of HIV was ranked among the top five notifiable infectious diseases (13). A series of prevention and treatment strategies have been implemented in China (14). In 2003, China launched the "four free and one care" policy for HIV (free treatment, free voluntary counseling and testing, free prevention of mother-tochild transmission, free schooling for AIDS orphans, and social care for HIV-infected patients) (15). In 2011, the "five expand six strengthen" policy was implemented in China to strengthen the national coverage, including HIV testing and surveillance, health education, blood administration and safety, prevention of mother-to-child transmission, antiretroviral therapies, as well as health care and social support (16). Since 2003, the National Free Antiretroviral Treatment Program (NFATP) has been implemented in China, despite the limited variety of free antiretroviral drugs. With the support of NFATP, the ART has undergone continuous evaluation and improvement (17). Over the past decades, China has made substantial achievements in the prevention and treatment of HIV. However, it remains a challenge to prevent and control HIV infections in China because of dynamic transportation, economic development, and social and cultural dynamics (16,18). In the past 20 years, the incidences of intravenous drug use, mother-to-child transmission, and blood transmission decreased substantially in China (19), but an increase in HIV infections can be Abbreviations: AIDS, Acquired immune deficiency syndrome; AAPC, Annual percentage changes; APC, Annual percentage changes; ART, Antiretroviral therapies; HIV, Human immunodeficiency virus; INI, Integrase inhibitor; MSM, Men who sex with men; NFATP, The National Free Antiretroviral Treatment Program; NNRTIs, Non-Nucleoside reverse transcriptase inhibitors; NRTI, Nucleoside reverse transcriptase inhibitor.  found in the elderly population (14,20) and young students (21). Sexual transmission remains the primary route of HIV transmission in China (22), but the incidences of homosexual transmission are growing in recent years (23)(24)(25). Furthermore, the geographical distribution of HIV infections varies in different provinces and regions in China (14). For instance, homosexual transmission in a province in northeast China accounted for the majority (69%) of HIV infections from 2011 to 2012 (26). Another epidemiological feature is that 77.1% of HIV infections in Southeastern China were found in patients aged 19-50 years (27). Taken together, it remains important to explore local epidemiological features to control HIV infections.
Hunan province, located in South-Central China, is known for its large population and is one of the most popular tourist destinations in China. Since the first report of HIV infection in Hunan Province in 1992, there is a growing trend of HIV incidences. However, no report has published epidemiological and clinical features of HIV-infected patients in Hunan province. Here, we analyzed a large-scale cohort of 32,419 HIV-infected patients who were diagnosed from 2003 to 2018 in Hunan province. Our study will provide the first comprehensive survey of the temporal trend, spatial distribution, and population characteristicsof HIV infection, virological responses, and survival status of HIV-infected patients who received standard ART. This study will shed light on the temporal trend of HIV infections in a high-risk population for better management and prevention of HIV infections in Hunan province.

Definitions
Failure of immune reconstitution was defined by the persistent CD4 level <100 cells/µl (28). As described previously (29), HIV-infected patients experienced the status of poor immune reconstruction if two conditions were fulfilled: (i) CD4+ T-cell counts < 350 cells/µl; and (ii) CD4+ T-cell increases < 100 cells/µl in those patients with HIV RNA <50 copies/ml for more than 1 year. Advanced HIV disease was defined as presenting for HIV care with CD4+ T-cell counts < 200 cells/µL or WHO stage 3/4 conditions.

Statistical Analysis
Categorical variables were presented as frequencies and percentages, while continuous variables were analyzed by mean and standard deviations. To analyze the temporal trend of new HIV infections per year, we used Joinpoint regression models (https://surveillance.cancer.gov/joinpoint/) to calculate the annual percentage changes (APC) as well as the trend test of APC. The grid search method was used to determine the joinpoint, and permutation tests were used to select the optimal model. Paired-t-tests were used to detect differences in CD4 levels before and after treatment. Chi-square tests were applied to compare the proportion of virological responses in patients receiving different ART. Welch's ANOVA methods were used to compare the means of multiple groups, while Games-Howell tests were used for pairwise comparisons between groups. P-values of multiple comparisons were corrected by the Holm method. The survival rate was estimated by the Kaplan-Meier method, while log-rank tests were used to evaluate any difference. Our statistical analyses used the pairwise deletion approach to handle missing data. All statistical analyses were descriptive, and no random sampling was conducted. A statistical significance was considered when a p-value was below 0.05. We performed statistical analyses using the Joinpoint Regression Program 4.9.0.0, R x64 4.1.0, and GraphPad prism 8.0.1.

RESULTS
From 2003 to 2018, a total of 34,297 HIV-infected patients were treated at the designated hospitals in Hunan province. Patient information is summarized in Table 1. Gender disparity was observed with a large proportion of male patients (N = 25,338, 73.9%). The number of HIV infections in both males and females increased over time, whereas the increasing numbers grew faster in males (Figure 1A). At the time of their first HIV-positive diagnosis, the youngest and oldest patients were 7 and 95 years of age, respectively. The median age at the first diagnosis of HIV infection was 46 years old. Among all age groups, patients aged ≥50 years accounted for the largest proportion at all times, the number of HIV-infected patients aged <30 years increased steadily from 2010 to 2018 ( Figure 1B).  (Figure 2A). In contrast, the annual percentage change from 2004 to 2018 (APC 2014−2018 ) was 6.62%, indicating a decreasing growth rate from 2014 to 2018 ( Table 2). Although new HIV cases seemed to increase in both males and females, the number and the growth trend of HIV cases in males were significantly higher (Figure 2A). As shown in Figure 2B, the incidences of HIV new cases increased in all age groups (<30 years, 30-39 years, 40-49 years, ≥50 years). A significant increase in HIV infections was identified among patients <30 years (AAPC 2013−2018 : 60.9%) ( Figure 2B).

The Spatial Disparity of HIV-Infected Patients
We analyzed the spatial disparity of HIV-infected patients using geographical distribution maps (Figure 3). As shown in Table 1 Figure 3A). Western Hunan had the second-lowest number of HIV infections, but its case fatality rate was the highest (Figure 3B). Southern Hunan had the most cases of HIV infections and HIV-associated deaths. The growth rate of HIV infections in the above five regions varied (Figure 2D), but the growth trend slowed down or decreased from 2014 to 2018 (Supplementary Table S1).

Dynamic Changes in HIV Transmission Routes
From 2003 to 2005, intravenous drug use and blood transmission were the major transmission routes (>50%) of HIV infections  Table 2).

ART Regimens
The ART regimen of two nucleoside reverse transcriptase inhibitors (NRTIs) plus one non-nucleoside reverse transcriptase inhibitor (NNRTI) was administered to a majority of HIV-infected patients (90.4%, N = 30,995). The most frequently prescribed NRTI backbones were a fixed combination of tenofovir and lamivudine (60.3%, N = 20,657  Figure S1). Although the number of advanced HIV disease increased over time, the proportion of advanced HIV disease from 2003 to 2018 showed decreasing patterns (Figure 4). In 2018, the proportion of advanced HIV disease was 31.9% in young patients aged <30 years (Supplementary Table S2). The median CD4+ T-cell counts at baseline were 212 cells/µl. CD4+ T-cell counts differed significantly in different age groups ( Figure 5A) and elderly patients had lower CD4+ T-cell counts at baseline (elderly patients: 191.0 cells/µl, young patients: 294.6 cells/µl, p-value < 0.0001). As shown in Figure 5B, baseline CD4+ T-cell counts were higher in homosexual male patients (297.47 cells/µl), followed by heterosexual females (225.36 cells/µl), and heterosexual males (209.42 cells/µl). After laboratory-confirmed diagnosis, HIV-positive patients received antiviral treatment with regular follow-up of CD4 counts every 6 months. First, CD4 counts increased significantly by comparing serum levels before treatment initiation and after 6-month treatment (252.9 vs. 349.1 cells/µl, p-value < 0.0001). Second, an average increase in CD4 levels of 105.5 cells/µl was observed among those patients who received treatment for 0.5-1 year. A significant increase in CD4 counts (452.2 cells/µl) was also observed among those patients treated for >5 years ( Table 3). Third, higher levels of CD4 counts at baseline were associated with a higher increase in CD4 counts ( Figure 5C) and CD4/CD8 ratio (Figure 5D; Supplementary Table S3). After >5 years of treatment, except for the patient group with baseline CD4 levels below 200 cells/µl, CD4 counts in other groups reached >500 cells/µl (Figure 5C). Those patients with baseline CD4 levels >200 cells/µl experienced the recovery of the CD4/CD8 ratio to 0.75 after >5 years of antiviral treatment (Figure 5D).

DISCUSSION
Based on a large-scale cohort of 34,297 HIV-infected patients, our study presents the first survey of HIV infections (2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015)(2016)(2017)(2018) in Hunan province. Compared with other provinces in China, the HIV epidemic in Hunan province is not considered severe (30). Although there seems a growing number of HIV incidences, the overall growth rate in Hunan province has slowed down in recent years, implying the effectiveness of HIV prevention and control strategies. Most HIV infections in Hunan province are diagnosed in male patients, in agreement with other studies conducted from other provinces in China (27,31). Our study finds that the majority of HIV infections in Hunan province are elderly patients aged ≥50 years, which is agreement with other national surveys (32)(33)(34). However, young males are considered the key population for HIV prevention given the increasing proportion of homosexual transmission. Although the mortality rate in young patients aged <30 years was relatively low, a significant increase in HIV infections can be traced to young populations, especially those high school and college students (21,35).
Due to the long incubation period from HIV infection to the onset of AIDS and related symptoms, early HIV diagnosis remains difficult in the absence of routine screening (17). Our study showed that the proportion of HIV-infected patients with CD4+ T cell counts < 200 cells/µl at baseline was 42.8% in 2018 in Hunan Province, which was relatively higher than that in other regions (36,37). In response to the national prevention strategies, free HIV testing services are currently available in Hunan to reduce the risk of advanced HIV disease. Early intervention, early detection, early control, and early treatment are strongly advocated in China, leading to better management of HIV infections in the future.
We observed a dynamic change in HIV transmission routes in the past decade. Before 2005, blood transmission and intravenous drug use are the main routes of transmission in China (16). To date, sexual transmission has become the predominant route of HIV transmission (22). Nevertheless, there is a growing trend of homosexual transmission across the country (25). In agreement with previous studies (22,25), we found that homosexual transmission was more likely to occur in young males in Hunan province. We also observed a higher level of baseline CD4+ T-cell counts in After treatment for more than 5 years, patients with different baseline CD4 counts were diagnosed with the follow-up of CD4 counts (C) and CD4/CD8 ratio (D). Only statistically significant groups were labeled.  both heterosexual and homosexual transmissions among highrisk populations.
Our study observed a difference in the case fatality rate among five geographical regions in Hunan province (Figure 3). The number of HIV infections was most in Southern Hunan, but the HIV-associated fatality rate was the highest in Western Hunan. This regional disparity might be explained by several reasons. (i) Economic and medical conditions are discrepant in different regions. (ii) Social stigma in different regions may play a role in HIV prevention and treatment. A recent study found that college students with higher education levels still hold negative attitudes toward HIV-infected patients (45). Previous studies suggest that HIV-related stigma and lack of social support have the potential to harm the health and wellbeing of HIV-infected patients (46,47). (iii) HIV is known for its high genetic diversity (48), and the distribution of circulating HIV subtypes and strains exhibit regional differences (49)(50)(51)(52). Further studies need to address the impact of diversified anti-HIV strategies and HIV strains on HIV prevention and treatment in different regions.
It is known that wide applications of HIV prevention and treatment strategies have reduced HIV-associated mortality and morbidity, not only in China but also in many other countries (53). We estimated 70.0% of the overall 10-year survival rate of treated patients using a large-scale cohort in Hunan province. Compared with other patient groups, elderly males in Western Hunan had a lower survival rate (Figure 6). It is known that HIV infection can impair the human immune system (54), and the elderly are more vulnerable to infectious diseases because aging can affect the innate and adaptive immune system (55). As expected, we observed an age-related decrease in CD4 levels at baseline. In agreement with previous studies (56, 57), we found that baseline CD4 levels were associated with the recovery of CD4 levels and CD4/CD8 after ART. Furthermore, elderly patients are unlikely to achieve the same virological and immune responses as young patients, probably due to the low CD4 levels at baseline and relatively poor immune function (58).
There are limitations to our study. First, our database included the majority (>70%) of laboratory-confirmed HIV-infected patients in Hunan province, but this official database does not include undiagnosed cases or those immigrant patients who were not designated to local health facilities in Hunan province. Second, our database only included HIV-associated factors, but not other factors such as ethnicity, occupation, degree of education, marital status, or traveling history. Therefore, our analysis cannot address all potential risk factors associated with HIV prevention and treatment. Third, our retrospective study could not reveal clinical efficacies of ART because HIV-infected patients were not randomized and treatment switch is often considered at different timepoints in clinical practice. Fourth, our study only focused on HIV epidemiology in Hunan Province, and future studies need to report HIV epidemiology from a global perspective.

CONCLUSION
This study reveals epidemiological and clinical characteristics of HIV-infected patients in Hunan province, shedding light on the focus of HIV prevention and treatment in certain high-risk populations and geographical regions. Although the overall growth rate of new HIV cases slows down in Hunan province, a special focus should be taken on elderly males who were infected mainly by heterosexually transmission. Furthermore, a high proportion of advanced HIV disease indicates the importance of HIV routine testing and surveillance. Better management strategies are still needed to effectively control the spread of HIV infections from a regional and global perspective.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available after the approval of the Ethics Committees.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Ethics Committees of The First Hospital of Changsha (Approval ID: 202160). Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.

AUTHOR CONTRIBUTIONS
TY performed statistical analyses and drafted the manuscript. PZ, TYH, and ED contributed with data interpretation and discussions of the manuscript. JH, JZ, and FZ performed data acquisition. XYH and FZ supervised the study. GL obtained funding and revised the manuscript. All authors contributed to the final article. All authors contributed to the article and approved the submitted version.